Differential expression of matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in non-melanoma skin cancer: implications for tumour progression

AIMS: To investigate the expression of matrix metalloproteinase (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in non-melanoma skin cancer (NMSC) and to compare their expression between different tumour types and with clinicopathological factors. METHODS AND RESULTS: A study of 11 normal skin, 29 Bowen's disease (BD), 40 squamous cell carcinoma (SCC) and 38 basal cell carcinoma (BCC) samples for MMP-2, MMP-9, TIMP-1 and TIMP-2 expression was carried out using immunohistochemistry and in situ hybridization. The expression of all metalloproteinases was greater in tumours than in normal skin. MMP-2 and MMP-9 expression was more extensive in the stroma of SCC than of BCC or BD. TIMP-1 expression was greater in the stroma of BCC than of SCC or BD and TIMP-2 expression was greater in the stroma of SCC than of BD. There was a correlation between increased metalloproteinase expression and depth of lesion (MMP-2 and TIMP-2), inflammation (MMP-2, MMP-9, TIMP-1 and TIMP-2) and microvessel density (MMP-2, MMP-9 and TIMP-2). CONCLUSIONS: MMP-2, MMP-9, TIMP-1 and TIMP-2 play an important role in the pathogenesis of non-melanoma skin cancer, but differ significantly in their expression levels between the tumour types examined. The immunoexpression of these proteins may be useful indicators of cutaneous cancer invasion and progression.     

Upregulation of tissue inhibitor of metalloproteinases (TIMP)-2 promotes matrix metalloproteinase (MMP)-2 activation and cell invasion in a human glioblastoma cell line

Local invasiveness is a characteristic feature of glioblastoma that makes surgical resection nearly impossible and accounts in large part for its poor prognosis. To identify mechanisms underlying glioblastoma invasion and motility, we used Transwell invasion chambers to select for a more potently invasive subpopulation of U87MG human glioblastoma cells. The stable population of tumor cells (U87-C1) obtained through this in vitro selection process were three times more invasive than parental U87MG cells and demonstrated faster monolayer wound healing and enhanced radial motility from cell spheroids. This enhanced invasiveness was associated with an 80% increase in matrix metalloproteinase 2 (MMP-2) activation. No differences in expression levels of pro-MMP-2, membrane-type matrix metalloproteinase I (MT1-MMP), or integrin alphavbeta3 (mediators of MMP-2 activation) were detected. However, U87-C1 cells exhibited two-fold elevation of tissue inhibitor of metalloproteinases (TIMP)-2 mRNA and protein relative to parental cells. Exogenous addition of comparable levels of purified TIMP-2 to parental U87MG cells increased MMP-2 activation and invasion. Similarly, U87MG cells engineered to overexpress TIMP-2 at the same levels as U87-C1 cells also demonstrated increased MMP-2 activation, indicating that an increase in physiological levels of TIMP-2 can promote MMP-2 activation and invasion in glioblastoma cells. However, exogenous administration or recombinant overexpression of higher amounts of TIMP-2 in U87MG cells resulted in inhibition of MMP-2 activation. These results demonstrate that the complex balance between TIMP-2 and MMP-2 is a critical determinant of glioblastoma invasion, and indicate that increasing TIMP-2 in glioblastoma patients may potentially cause adverse effects, particularly in tumors containing high levels of MT1-MMP and MMP-2.     

Quantitative differences in matrix metalloproteinase (MMP)-2, but not in MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 or TIMP-2, in seminal plasma of normozoospermic and azoospermic patients

BACKGROUND: Matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), have been detected in reproductive tissues and seminal plasma. The purpose of this study was to quantify MMP-2, MMP-9, TIMP-1 and TIMP-2 in human seminal plasma and to evaluate their association with sperm. METHODS: Seminal plasma was analysed using ELISA assays for all four analytes in 12 normozoospermic and 12 azoospermic patients and then for MMP-2 only in another 114 men with azoospermia (n = 16), after vasectomy (n = 20) and with sperm counts within the following ranges: 0.3-19 x 10(6)/ml (n = 20), 20-23 x 10(6)/ml (n = 11), 49-57 x 10(6)/ml (n = 12), 96-110 x 10(6)/ml (n = 12), 139-161 x 10(6)/ml (n = 12) and 215-346 x 10(6)/ml (n = 11). Additional zymographic analyses using SDS-PAGE were performed. RESULTS: All investigated MMPs and TIMPs were detected. MMP-9, TIMP-1 and TIMP-2 were not significantly different in normozoospermia and azoospermia. Only the MMP-2 concentration was significantly decreased in azoospermic compared with normozoospermic patients (mean +/- SD: 650.6 +/- 288.9 versus 1677 +/- 910.4 ng/ml respectively; P = 0.0002) and significantly correlated with the number of sperm (r = 0.54; P < 0.0001). CONCLUSION: MMP-2 in seminal plasma was strongly correlated to the sperm count in a linear fashion. Its origin and potential function remain to be elucidated.     

Reduced expression of tissue inhibitor of metalloproteinase in nodal metastasis of stomach cancer.

The matrix metalloproteinases (MMPs) have been associated with tumor cell invasion and metastasis of human cancers by mediating the degradation of extracellular matrix components. Therefore, these enzymes and their inhibitor (TIMP-2) constitute promising targets in the development of anticancer therapies. In order to investigate the correlation between expressions of TIMP-2, MMPs and clinical outcome, immunohistochemical staining of MMP-2, MMP-9, and TIMP-2 were performed on paraffin-embedded tissue sections of 15 early gastric cancers (EGC) and 15 advanced gastric carcinomas (AGC) without nodal metastasis and 15 AGC with nodal metastasis (AGCn+). MMP-2 and MMP-9 were expressed in neoplastic cell plasma membrane in 83.3% and 88% of cases of AGC, respectively with inter-tumoral variability of staining intensity. MMP-2 and MMP-9 staining were not correlated with presence of nodal metastasis or degree of invasion depth at the time of diagnosis (p>0.05). The immunoreactivity of TIMP-2 was detected in the peri-tumoral stroma. Residual benign stomach tissue showed no or weak immunoreactivity for TIMP-2 staining. Among AGC, neoplasms with diffuse and strong TIMP-2 staining have less frequent metastasis (28.6%) than cases with focal and weak (68.8%) (p<0.05). Early gastric cancer revealed diffuse and strong TIMP-2 expressions. We conclude that clinical outcome such as depth of invasion or metastasis is more closely related to the expression of TIMP-2 than the corresponding MMPs.     

组织金属蛋白酶抑制剂TIMP-2基因转染对胃癌细胞体内外侵袭能力的影响

目的了解ＴＩＭＰ２基因转染对胃癌细胞侵袭行为的影响，进行阻断肿瘤细胞侵袭转移的筛选尝试。方法采取脂质体将外源基因ＴＩＭＰ２导入人胃癌细胞系ＳＧＣ７９０１，经体外Ｂｏｙｄｅｎ小室测定肿瘤细胞侵袭能力与体内肾包膜下瘤组织种植侵袭模型对比分析，观察Ⅳ型胶原酶前体（ＭＴＭＭＰ）与基底膜Ⅳ型胶原免疫组化改变。结果发现转染ＴＩＭＰ２基因的胃癌细胞其表达ＭＴＭＭＰ的能力降低，成瘤时间延长，成瘤率降低，体外与体内的侵袭能力受到抑制。结论ＴＩＭＰ２基因转染可使胃癌细胞的侵袭表型得到下调。     

Prognostic significance of matrix metalloproteinase 2 and tissue inhibitor of metalloproteinase 2 expression in prostate cancer.

Matrix metalloproteinases (MMPs) are proteolytic enzymes capable of degrading the structural support network for normal and malignant cells, promoting neoplastic cell invasion and metastasis. Tissue inhibitors of metalloproteinases (TIMPs) maintain connective tissue integrity by modulating MMP activity. Formalin-fixed paraffin-embedded tissue sections from 138 prostatic adenocarcinomas (PACs) were immunostained by a combined automated/manual method using monoclonal antibodies against MMP2 and TIMP2. Immunoreactivity was semiquantitatively scored based on stain intensity and distribution, and results were correlated with Gleason grade, pathologic stage, ploidy status, and disease recurrence. One hundred five of 138 (76%) and 113/138 (82%) PACs expressed MMP2 and TIMP2, respectively. Co-expression was observed in 94/138 (68%) of PACs (P =.01), correlated with advanced tumor stage (P =.05), and tended to be associated with disease recurrent cases (P =.07). TIMP2 expression individually correlated with advanced tumor stage (P =.04) and reached near significance with disease recurrence (P =.06). MMP2 expression was also more frequent in recurrent PACs, although this value did not reach statistical significance (P =.07). However, on multivariate analysis, only pathologic stage (P =.009) and ploidy status (P =.03) independently predicted disease recurrence. In conclusion, MMP2 and TIMP2 are co-expressed in a majority of PACs and correlate with prognostic variables. Interestingly, contrary to the previously documented anti-tumor effects of TIMPs, TIMP2 expression appears to have a tumor-promoting role in PACs and warrants further investigation.     

Induced sputum-retrieved matrix metalloproteinase 9 and tissue metalloproteinase inhibitor 1 in granulomatous diseases

Matrix metalloproteinases (MMPs) capable of degrading various components of connective tissue matrices, and tissue inhibitor metalloproteinases (TIMPs) are considered important in lung parenchymal remodeling and repair processes in pulmonary diseases. Induced sputum (IS) is a reliable noninvasive method to investigate pathogenesis, pathophysiology and treatment of lung disease. This study was designed to determine whether IS-MMP9/TIMP1 levels demonstrate lung parenchymal remodeling in sarcoidosis (SA) and Crohn's disease (CRD) patients. Sputum was induced and processed conventionally in 13 SA patients, 18 CRD patients and 9 controls. Two-hundred cells were counted on Giemsa-stained cytopreps, and T lymphocytes subsets (CD4 = T helper and CD8 = T suppressor cytotoxic cells) were analysed by FACS using monoclonal antibodies.MMP-9 and TIMP-1 were measured using commercial ELISA kits. MMP-9 concentrations, but not those of TIMP-1, were significantly greater in the sputum supernatant in SA and CRD patients compared to controls (P = 0.018 and P = 0.0019, respectively). The molar ratio, MMP-9/TIMP-1, was significantly higher in SA and CRD patients compared to controls (P = 0.008 and P = 0.024, respectively). Gelatinase species having a molecular weight similar to that of MMP-9 were demonstrated by zymographic analysis. MMP-9 levels were highly correlated with the CD4/CD8 ratio and DLCO capacity in SA but less in CRD patients. MMP-9 levels in IS provide a sensitive marker for pulmonary damage.     

Matrix metalloproteinase 2 (MMP-2) and their tissue inhibitor 2 (TIMP-2) in gastric cancer patients

BackgroundMatrix metalloproteinase 2 (MMP-2) is able to degrade type IV collagen and its activity is mostly regulated by tissue inhibitor of matrix metalloproteinase 2 (TIMP-2). These proteins might play a role in tumor progression, including gastric cancer (GC).MethodsThe study included 108 individuals, GC patients and healthy subjects. Serum levels of all analyzed markers were evaluated by the immunological methods, while immunohistochemistry was used to assess the expression of these proteins in GC, interstitial inflammatory cells and normal tissues.ResultsThe percentage of positive reactions of MMP-2 and TIMP-2 was higher in GC and inflammatory cells compared to normal tissue, while serum levels of these proteins were statistically lower in GC patients in comparison to healthy subjects. There was a significant positive correlation between TIMP-2 immunoreactivity in inflammatory cells and the presence of lymph node metastasis. Area under ROC curve (AUC) for TIMP-2 was higher than MMP-2, while serum MMP-2 was an independent prognostic factor of GC patients' survival.ConclusionOur findings suggest that TIMP-2 seems to be a predictor of tumor progression, especially for nodal involvement, whereas serum MMP-2 might be useful as an independent prognostic factor of patients' survival.     

Tissue inhibitor of metalloproteinases (TIMP)-1, -2 and -3 in human endometrium during the menstrual cycle

The extensive remodelling of the human endometrium throughout the menstrual cycle is accompanied by changes in production of matrix metalloproteinases, the activity of which can be inhibited by specific tissue inhibitors or by tissue inhibitors of metalloproteinases (TIMP)s with a 1:1 stoichiometry. This study immunolocalized TIMP-1, TIMP-2 and TIMP-3 in dated normal human endometrium across the menstrual cycle and examined cultured endometrial cells for their production. All three TIMPs were present in the major cellular compartments, luminal epithelium, glands, stroma, endothelial cells and vascular smooth muscle cells with the most intense immunoreactivity in the luminal epithelium. TIMP-1 and -3 were lower in the mid-to-late proliferative phase with a nadir of TIMP-3 particularly in the late proliferative phase. Decidualized stromal cells stained strongly positive for TIMP-1, -2 and -3. Cells of haematopoietic origin never stained. Monensin treatment of tissue resulted in accumulation of TIMPs in all cellular compartments but particularly of TIMP-1 in epithelium. Cultured endometrial stromal cells released more TIMP-1 than TIMP-2 or TIMP-3 into culture medium and all were increased following decidualization in vitro. Epithelial cells in culture produced less TIMPs than stromal cells, and only a few epithelial cells in each culture were immunopositive for TIMP-1. The ubiquitous distribution of TIMPs implicates them in maintenance of endometrial integrity, with changes in the matrix metalloproteinases without concomitant changes in TIMPs determining endometrial matrix degradation.      

The expression and localization of neutral endopeptidase 24.11/CD10 in human gestational trophoblastic diseases

Neutral endopeptidase 24.11 (NEP)/CD10 is a cell-surface peptidase that hydrolyzes various bioactive peptides. NEP is distributed in both normal and neoplastic cells and plays a functional role by modulating cellular responses to peptide substrates. Recently, NEP has been shown to be expressed in normal placental trophoblasts, suggesting its physiological role during pregnancy. In the present study, we investigated the expression of NEP in hyperplastic and anaplastic trophoblasts in gestational trophoblastic diseases (GTDs). Flow cytometric analysis demonstrated that NEP was expressed in all choriocarcinoma cell lines examined. The NEP enzyme activity in these cell lines correlated with cell-surface protein levels and was abolished by the NEP inhibitor phosphoramidon. On immunoblot analysis, NEP protein was detected in both hydatidiform mole and choriocarcinoma tissues as a double band of 95 and 100 kDa similar to that of the normal placental tissues. Immunohistochemical analysis revealed that NEP was present on syncytiotrophoblasts, while no or very faint NEP immunoreactivity was observed on cytotrophoblasts in the normal placenta. Similarly, NEP in hydatidiform mole and invasive mole was localized on the membrane of syncytiotrophoblasts, but not on hyperplastic cytotrophoblasts. In contrast, in choriocarcinoma, NEP was highly expressed not only on syncytiotrophoblastic cells but also on invading anaplastic cytotrophoblasts. In addition, NEP was also expressed on intermediate trophoblasts in placental site trophoblastic tumors. In summary, this is the first study demonstrating the expression of NEP/CD10 in GTDs. The differential localization of NEP among various trophoblastic tumors suggests that NEP may play a functional role in the regulation of trophoblast transformation and human chorionic gonadotropin secretion.     

PCNA和Caspase-3在妊娠滋养细胞疾病中的表达及意义

目的研究PCNA和Caspase-3在妊娠滋养细胞疾病中的表达变化及意义,为妊娠滋养细胞疾病早期诊断、预后判断提供理论依据。方法收集滋养细胞疾病标本50例,其中葡萄胎10例(均为完全性葡萄胎),侵蚀性葡萄胎20例,绒毛膜癌20例。正常早孕绒毛标本10例为对照组。应用免疫组织化学技术检测PCNA和Caspase-3在早孕绒毛、葡萄胎、侵蚀性葡萄胎、绒毛膜癌组织中的表达情况。结果 PCNA在正常早孕绒毛、葡萄胎、侵蚀性葡萄胎和绒癌中的表达分别为10.24%、20.70%、50.92%、53.65%,各组差异有统计学意义(P0.05)。Caspase-3在正常早孕绒毛、葡萄胎、侵蚀性葡萄胎和绒癌中的表达先升高又降低,分别为1.25%、2.60%、6.40%、1.90%,各组差异有统计学意义(P0.05)。结论 PCNA及Caspase-3表达异常,增殖/凋亡失衡是引起GTT的重要因素。     

彩色多普勒超声诊断子宫恶性滋养细胞肿瘤的探讨

目的 评价彩色多普勒血流显像(CDFI)及脉冲多普勒(PD)检测恶性滋养细胞肿瘤的早期诊断疗效观察及判断疾病转归的应用价值。方法 应用CDFI及PD检测恶性滋养细胞肿瘤患者32例,并以正常妊娠20例作对照。结果 CDFI恶性滋养叶肿瘤表现为丰富而敏感的彩色血流,PD为低速的动静脉血流。正常妊娠为不甚敏感的点状或多条状的不规则血流,PD为单相或双相的低速动脉血流。阻力指数(RI)显著性差异(P<0.01)。结论 CDFI和PD检测可做为一种新的方法,不仅可对滋养叶细胞肿瘤作出早期诊断,亦可用于观察化疗效果,判断疾病的转归。     

基质金属蛋白酶及其抑制物在高碳酸血症大鼠肺组织中的表达

目的:观察单纯高碳酸血症大鼠模型的血清中性粒细胞蛋白酶(NE)、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)和金属蛋白酶组织抑制剂(TIMP-1)的活性,以及它们在肺组织中的表达,探讨高浓度二氧化碳(CO2)引起肺组织损伤的机制。方法:雄性Wistar大鼠40只,随机分为正常对照组(A组,20只大鼠)、高碳酸血症组(B组,20只大鼠)。B组大鼠置于高CO2舱中,并向舱中匀速输入6%CO2混合气体(6%CO2,21%O2,73%N2),每天7h,连续4周,完成大鼠高碳酸血症模型的复制。用ELISA法测定大鼠血清MMP-2、MMP-9、TIMP-1活性及NE活性;弹力纤维染色标本进行弹性纤维相对含量的测定;免疫组化方法检测肺组织中MMP-2、MMP-9及TIMP-1的表达及光镜下进行病理学观察。结果:B组大鼠血清MMP-2、MMP-9、TIMP-1活性与A组之间并无显著差异(P0.05);B组大鼠NE活性明显高于A组(P0.01);光镜下可见B组大鼠肺泡壁明显增厚,肺微血管内皮细胞损伤,小静脉扩张,血栓形成;小动脉内皮细胞肿胀,管腔狭窄,血管周围有袖套状水肿区;并可见小灶性肺实质出血;A组大鼠弹力纤维呈束状,连续无断裂;B组大鼠可见弹力纤维断裂降解,且肺组织中弹力纤维的含量较A组明显减少(P0.01);B组大鼠肺组织中MMP-2的表达明显低于A组(P0.01);而肺组织中MMP-9及TIMP-1的表达B组大鼠明显高于A组(P0.01)。结论:通过复制出常压、常氧、单纯高碳酸血症动物模型;单纯的PaCO2增高对肺动脉压无明显影响;高浓度CO2可以使NE活性明显增高,使弹性蛋白的降解增多,肺组织弹性纤维断裂降解,数量减少;在单纯高碳酸血症动物模型中,MMP-2的表达明显降低,MMP-9及TIMP-1在肺组织中表达明显增高,导致肺损伤。     

MM P-2/TI MP-2及VEG F在滋养细胞疾病中的表达及意义

目的 检测不同滋养细胞疾病组织中MMP-2/TIMP-2及VEGF的表达情况,观察比较它们的表达特点,分析与滋养细胞疾病发生、发展的相关性.方法 收集30例正常绒毛;40例葡萄胎组织,30例手术切除的恶性葡萄胎组织,采用单克隆抗体免疫组化SP法对其染色,光镜下分别观察以上不同组织中MMP-2、TIMP-2、VEGF的阳性表达情况.结果 (1)从正常绒毛→良性葡萄胎→恶性葡萄胎,MMP-2的阳性率有升高趋势,差异有统计学意义(P＜0.05).VEGF的表达与MMP-2正相关.(2)TIMP-2的阳性表达在滋养细胞疾病内部不同分组间两两比较,差异均无统计学意义(P＞0.05).(3)MMP-2/TIMP-2组合同时阴性表达在良性葡萄胎中占65%(26/40),在恶性葡萄胎中占16.67%(5/30),两者比较有显著性差异(P＜0.01).MMP-2阳性表达伴TIMP-2阴性表达在良性葡萄胎中占2.5%(1/40),在恶性葡萄胎中占56.67%(17/30),两者比较有显著性差异(P＜0.01).结论 (1)MMP-2和VEGF共同参与了滋养细胞疾病的发生和发展.(2)MMP-2/TIMP-2两者联合检测对鉴别良、恶性滋养细胞疾病有重要价值.     

Immunohistochemistry and other ancillary techniques in the diagnosis of gestational trophoblastic diseases

Gestational trophoblastic disease (GTD) encompasses entities ranging from ubiquitous hydatidiform moles to rare neoplastic gestational trophoblastic tumors. In practice, the histological diagnosis of GTD continues to have significant diagnostic inaccuracy with marked inter- and intra-observer variability, even among expert pathologists. Studies in correlation with genotypic evidence have confirmed a lack of accuracy in diagnosis of hydatidiform moles using histology alone. Applications of new immunohistochemical markers and molecular techniques have significantly enhanced the diagnostic precision of various GTDs in recent years. p57 Immunohistochemistry is a highly useful marker in confirming complete hydatidiform mole. PCR-based DNA genotyping has emerged as a powerful diagnostic measure to precisely classify both complete and partial hydatidiform moles. With acquisition of molecular diagnostic capabilities at most medical centers, these ancillary techniques have been increasingly integrated into the routine diagnostic workup of GTD. We propose an algorithmic approach combining histology and these ancillary tests to provide the best diagnostic practice possible. Under this algorithm, all cases with histological suspicion for complete mole are subject to p57 immunohistochemical confirmation, and all cases with histological suspicion for partial mole undergo DNA genotyping workup. Beyond hydatidiform mole, recognition of gestational trophoblastic tumors requires a high index of suspicion and application of immunohistochemical markers of trophoblast is helpful to accurately diagnose these rare tumors.     

p53在妊娠滋养细胞和肿瘤中的表达

目的：分析Ｐ５３过度表达与葡萄胎、恶性葡萄胎（恶葡）、绒毛膜癌（绒癌）的恶性程度及预后关系。方法：采用单克隆抗体免疫组化技术，对正常胎盘绒志及妊娠滋养细胞肿瘤组织，进行肿瘤抑制基因Ｐ５３表达检测。其中包括早期妊娠１０例，中期妊娠１０例，晚期妊娠１３例，葡萄胎４０例，恶葡９例，绒癌２０例；结果：正常胎盘Ｐ５３异常表达阴性，葡萄胎、恶葡、绒癌阳性表达率分别为４２．５％（１７／４０）、５５．５％（５／９